Sperm entry in mammalian oocytes triggers intracellular Ca2+ oscillations that initiate resumption of the meiotic cell cycle and subsequent activations. Here, we show that phospholipase C zeta 1 (PLCζ1) is the long-sought sperm-borne oocyte activation factor (SOAF). Plcz1 gene knockout (KO) mouse spermatozoa fail to induce Ca2+ changes in intracytoplasmic sperm injection (ICSI). In contrast to ICSI, Plcz1 KO spermatozoa induced atypical patterns of Ca2+ changes in normal fertilizations, and most of the fertilized oocytes ceased development at the 1–2-cell stage because of oocyte activation failure or polyspermy. We further discovered that both zona pellucida block to polyspermy (ZPBP) and plasma membrane block to polyspermy (PMBP) were delayed in oocytes fertilized with Plcz1 KO spermatozoa. With the observation that polyspermy is rare in astacin-like metalloendopeptidase (Astl) KO female oocytes that lack ZPBP, we conclude that PMPB plays more critical role than ZPBP in vivo. Finally, we obtained healthy pups from male mice carrying human infertile PLCZ1 mutation by single sperm ICSI supplemented with Plcz1 mRNA injection. These results suggest that mammalian spermatozoa have a primitive oocyte activation mechanism and that PLCζ1 is a SOAF that ensures oocyte activation steps for monospermic fertilization in mammals.

Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201–12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract–enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the “gold standard” to determine whether a gene’s function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarily conserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.

Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals. Here we show that large deletions with two sgRNAs as well as dsDNA-mediated point mutations are efficient in mouse embryonic stem cells (ESCs). The dsDNA-mediated gene knockins are also feasible in ESCs. Finally, we generated chimeric mice with biallelic mutant ESCs for a lethal gene, Dnajb13, and analyzed their phenotypes. Not only was the lethal phenotype of hydrocephalus suppressed, but we also found that Dnajb13 is required for sperm cilia formation. The combination of biallelic genome editing in ESCs and subsequent chimeric analysis provides a useful tool for rapid gene function analysis in the whole organism.

Flagella and cilia are critical cellular organelles that provide a means for cells to sense and progress through their environment. The central component of flagella and cilia is the axoneme, which comprises the "9+2" microtubule arrangement, dynein arms, radial spokes, and the nexin-dynein regulatory complex (N-DRC). Failure to properly assemble components of the axoneme leads to defective flagella and in humans leads to a collection of diseases referred to as ciliopathies. Ciliopathies can manifest as severe syndromic diseases that affect lung and kidney function, central nervous system development, bone formation, visceral organ organization, and reproduction. T-Complex-Associated-Testis-Expressed 1 (TCTE1) is an evolutionarily conserved axonemal protein present from Chlamydomonas (DRC5) to mammals that localizes to the N-DRC. Here, we show that mouse TCTE1 is testis-enriched in its expression, with its mRNA appearing in early round spermatids and protein localized to the flagellum. TCTE1 is 498 aa in length with a leucine rich repeat domain at the C terminus and is present in eukaryotes containing a flagellum. Knockout of Tcte1 results in male sterility because Tcte1-null spermatozoa show aberrant motility. Although the axoneme is structurally normal in Tcte1 mutant spermatozoa, Tcte1-null sperm demonstrate a significant decrease of ATP, which is used by dynein motors to generate the bending force of the flagellum. These data provide a link to defining the molecular intricacies required for axoneme function, sperm motility, and male fertility. male infertility | asthenozoospermia | glycolysis | mutant mouse | testis-specific gene F lagella are ancient, analogous cellular structures used for locomotion and as sensory organelles present in all three domains of life (bacteria, archaea, and eukaryotes). The advantages conferred by this organelle are highlighted by the flagella's apparent independent evolution in all three domains (1-3). Of all of the different flagella present among eukaryotes, flagella attached to gametes play a critical function in uniting gametes for fertilization and the perpetuation of a species. Mammalian spermatozoa have a specialized flagellum that contains a midpiece, principal piece, and end piece with the axoneme running along the entire length (4). The flagellum equips sperm with the capability to deliver half of the male's genetic material to the female gamete, the oocyte. In addition to flagella, eukaryotes contain another related structure called cilia. The defining feature of flagella and cilia is the axoneme, the "9+2" microtubule arraignment of two central pairs of microtubules surrounded by nine pairs of microtubule doublets (5). The microtubule motor dynein is anchored to the outer microtubules and responsible for generating the force required to produce the beating pattern of flagella and cilia (6). The force generated by dynein causes sliding of the microtubules among each other; however, the nexin complex anchors the microtubules in place. The nexin complex [or nexin-dynei...

SignificanceThe most abundant modification in mRNA is the N6-methylation of internal adenosines (m6A), but m6A’s physiological function is unknown for most mRNAs. Here we show that Casein Kinase 1 Delta mRNA (Ck1δ), coding for a critical kinase in the control of circadian rhythms, is regulated by m6A. When m6A is inhibited, the expression of two CK1δ isoforms, uncharacterized until now, increases due to enhanced translation. This increase in CK1δs leads to a slower clock because of increased phosphorylation of the clock protein PER2 at a key residue, leading to the stabilization of PER2 protein.

Sperm–egg fusion is the critical step in mammalian fertilization, and requires the interaction between IZUMO1 on the sperm surface and JUNO (also known as folate receptor (FR) 4 or IZUMO1R) on the egg surface. Whereas other FRs bind and uptake folates, JUNO binds IZUMO1 and establishes the cell–cell adhesion. However, the mechanism of IZUMO1 recognition by JUNO has remained elusive. Here we report the crystal structure of mouse JUNO, at 2.3 Å resolution. A structural comparison of JUNO with the FRs revealed that JUNO and the FRs have similar overall structures, but JUNO lacks the folate-binding pocket, thereby explaining the inability of JUNO to bind folate. Further complementation of Juno knockout eggs with mutant Juno messenger RNAs revealed that the conserved, surface-exposed tryptophan residue of JUNO is required for sperm binding and fertilization. Our structure-based in vivo functional analyses provide a framework towards a mechanistic understanding of mammalian gamete recognition.

Sperm–oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. Sperm membrane protein IZUMO1 and its counterpart oocyte membrane protein JUNO make a protein complex allowing sperm to interact with the oocyte, and subsequent sperm–oocyte fusion. Oocyte tetraspanin protein CD9 also contributes to sperm–oocyte fusion. However, the fusion process cannot be explained solely by these three essential factors. In this study, we focused on analyzing a testis-specific gene 4930451I11Rik and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in 4930451I11Rik knockout (KO) spermatozoa, the KO spermatozoa were unable to fuse with oocytes and the KO males were severely subfertile. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form. Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm–oocyte fusion. Thus, we renamed this TM form Fertilization Influencing Membrane Protein (FIMP). The mCherry-tagged FIMP TM form was localized to the sperm equatorial segment where the sperm–oocyte fusion event occurs. Thus, FIMP is a sperm-specific transmembrane protein that is necessary for the sperm–oocyte fusion process.

More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories.